Generation of functional human ipsc-derived pancreatic islets in co-culture with isogenic ipsc-derived vascular endothelial cells

Abstract

Diabetes is a clinical condition that affects millions of people worldwide, and is treated by insulin replacement therapies. New strategies to create scalable and compatible pancreatic islets containing insulin-producing beta cells are necessary as an alternative to limited supply of cadaveric islets or multiple exogenous insulin applications. Improvements are still necessary since many immature polyhormonal cells remain, and cannot attain a monohormonal state. During human development, pancreas co-develops with endothelium and shares signals, allowing for better maturation of beta cells, and this is not included in the current differentiation protocols. The organchip microfluidic devices allows dynamic co-culture of different cells, thus resembling in vivo physiology. Here the Inventors establish organ-chip models co-culturing human iPSC-derived pancreatic precursors with iPSC-derived endothelial cells to obtain more functional and monohormonal iPSC-derived beta cells.

1 . A method of generating functional induced pluripotent stem cell (iPSC) derived pancreatic islets (iIslets), comprising:
 co-culturing a quantity of iPSC derived vascular endothelial cells (iECs) and a quantity of iPSC derived islet progenitors for about 10-18 days to generate the functional iIslets comprising β-cells.   
     
     
         2 . The method of  claim 1 , wherein co-culturing comprises:
 plating a quantity of iPSC derived vascular endothelial cells (iECs) on MATRIGEL-coated plates and culturing in Phase IV EC media supplemented with Y27632;   plating a quantity of iPSC derived pancreatic islets (iIslets) on top of the quantity of iECs and either culturing in media comprising about ½ Phase IV iEC media and about ½ Phase VI islet media supplemented with Y-27632 for about 12-16 days, or culturing in Phase VI islet media (islet only condition) for about 12-16 days, to generate the functional iIslets comprising β-cells.   
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , further comprising generating the iECs by:
 plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL;   culturing the iPSC in MATRIGEL for about 2-4 days;   culturing in the presence of CHIR99021 for about 1-3 days to generate mesoderm;   culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 1-3 days to generate vascular progenitors;   culturing the vascular progenitors in the presence of VEGF and Y-27632 for about 3-8 days to generate endothelial cell (EC) progenitors.   
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , further comprising first generating the quantity of islet progenitors by:
 culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of Activin-A, CHIR99021 and Y-27632 for about 1-2 days;   culturing in the presence of Activin-A and FGF2 for about 1-3 days;   culturing in the presence of FGF10, CHIR99021 and Noggin for about 1-3 days, to generate posterior foregut cells;   culturing the posterior foregut cells in the presence of FGF10, Noggin, RA and SANT1 for about 3-5 days to generate pancreatic progenitors;   culturing the pancreatic progenitors in the presence of Noggin, EGF and Nicotinamide for about 3-5 days to generate pancreatic endocrine progenitors;   culturing the pancreatic endocrine progenitors in the presence of Noggin, T3 and Alk5i II for about 6-8 days to generate islet progenitors.   
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 ,
 wherein the pancreatic progenitors express PDX1+ and SOX9+, or   wherein the pancreatic endocrine progenitors are PDX1+ and NKX6.1+, or   wherein the iIslets express C-peptide, glucagon and NKX6.1+.   
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 2 , wherein, the expression of INS, UCN3, NGN3 and CHGA are upregulated in the β-cell that are produced in the islets only condition, as compared to β-cell that are produced without co-culturing with vascular endothelial cells or as compared to β-cell that were produced in a culture without the islets only condition. 
     
     
         12 . The method of  claim 1 ,
 wherein the β-cell increase insulin secretion when challenged with a high glucose concentration as compared to a basal glucose concentration, or   wherein the iPSC derived vascular endothelial cells (iECs) and iPSC derived islet progenitors are isogenic, or   wherein the iPSCs used to derive vascular endothelial cells (iECs) and iPSC used to derive islet progenitors are from the same iPSC cell line or from the same donor, or   wherein the iIslets are human iIslets.   
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . Induced pluripotent stem cell (iPSC) derived pancreatic islets (iIslets) produced by a method of  claim 1 . 
     
     
         17 . Induced pluripotent stem cell (iPSC) derived pancreatic islets (iIslets) expressing C-peptide, glucagon and NKX6.1+. 
     
     
         18 . The iIslets of  claim 16 , wherein the iIslets increase insulin secretion when challenged with a high glucose concentration as compared to a basal glucose concentration. 
     
     
         19 . A method of ameliorating or treating a metabolic disease, metabolic disorder or metabolic condition in a subject in need thereof, comprising:
 administering iIslets of  claim 16  to the subject in need thereof to ameliorate or treat the metabolic disease, metabolic disorder or metabolic condition.   
     
     
         20 . The method of  claim 19 , wherein the metabolic disease, metabolic disorder or metabolic condition is diabetes or insulin resistance. 
     
     
         21 . A method, comprising:
 culturing a quantity of induced pluripotent stem cells (iPSCs) in the presence of Activin-A, CHIR99021 and Y-27632 for about 1 day;   followed by culturing in the presence of Activin-A and FGF2 for about 2 days; and   followed by culturing in the presence of FGF10, CHIR99021 and Noggin for about 2 days, to generate posterior foregut cells.   
     
     
         22 . The method of  claim 21 , further comprising culturing the posterior foregut cells in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to generate pancreatic progenitors. 
     
     
         23 . The method of  claim 22 , wherein the pancreatic progenitors express PDX1 +  and SOX9 + . 
     
     
         24 . The method of  claim 21 , further comprising culturing the pancreatic progenitors in the presence of Noggin, EGF and Nicotinamide for about 4 days to generate pancreatic endocrine progenitors. 
     
     
         25 . The method of  claim 21 , wherein the pancreatic endocrine progenitors are PDX1 +  and NKX6.1 + . 
     
     
         26 . The method of  claim 24 , further comprising culturing the pancreatic endocrine progenitors in the presence of Noggin, T3 and Alk5i II for about 7 days to generate islet progenitors. 
     
     
         27 . The method of  claim 26 , further comprising culturing the generated islet progenitors in the presence of T3, Alk5i II, R428, and N-acetylcysteine (NAC) for about 14 days to generate mature islets. 
     
     
         28 . The method of  claim 27 , wherein the mature islets express C-peptide, glucagon and NKX6.1 + . 
     
     
         29 . The method of  claim 21 , further comprising:
 culturing the posterior foregut in the presence of FGF10, Noggin, RA and SANT1 for about 4 days to generate pancreatic progenitors;   followed by culturing the pancreatic progenitors in the presence of Noggin, EGF and Nicotinamide for about 4 days to generate pancreatic endocrine progenitors;   followed by culturing the pancreatic endocrine progenitors in the presence of Noggin, T3 and Alk5i II for about 7 days to generate islet progenitors; and   followed by culturing the generated islet progenitors in the presence of T3, Alk5i II, R428, and N-acetylcysteine (NAC) for about 14 days to generate mature islets.   
     
     
         30 . A quantity of mature islets made by the method of  claim 27 . 
     
     
         31 . A method, comprising:
 plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL;   culturing for about iPSC in MATRIGEL for about 3 days; and   followed by culturing in the presence of CHIR99021 to generate mesoderm.   
     
     
         32 . The method of  claim 31 , further comprising culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular progenitors. 
     
     
         33 . The method of  claim 32 , further comprising culturing the vascular progenitors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) progenitors. 
     
     
         34 . The method of  claim 33 , further comprising culturing the EC progenitors with VEGF for about 10 days to generate mature EC. 
     
     
         35 . The method of  claim 34 , wherein the mature EC express CD31 + , CD144 + , VEGF-A + , VEGFR2 + , and Ac-LDL. 
     
     
         36 . The method of  claim 31 , further comprising:
 culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular progenitors;   followed by culturing the vascular progenitors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) progenitors; and   followed by culturing the EC progenitors with VEGF for about 10 days to generate mature EC.   
     
     
         37 . A quantity of mature EC made by the method of  claim 34 . 
     
     
         38 . An assembly, comprising a quantity of mature islets and a quantity of mature EC, wherein the mature islets and the mature EC are isogenic. 
     
     
         39 . The assembly of  claim 38 ,
 wherein the quantity of the mature islets made by a process comprising co-culturing a quantity of iPSC derived vascular endothelial cells (iECs) and a quantity of iPSC derived islet progenitors for about 10-18 days to generate mature iIslets comprising β-cells, and   wherein the quantity of the mature ECs is made by a process comprising:
 plating a quantity of induced pluripotent stem cells (iPSCs) onto MATRIGEL; 
 culturing for about iPSC in MATRIGEL for about 3 days; 
 followed by culturing in the presence of CHIR99021 to generate mesoderm; 
 culturing the mesoderm in the presence of BMP4, FGF2, and VEGF for about 2 days to generate vascular progenitors; 
 culturing the vascular progenitors in the presence of VEGF and Y-27632 for about 7 days to generate endothelial cell (EC) progenitors; and 
 culturing the EC progenitors with VEGF for about 10 days to generate mature EC. 
   
     
     
         40 . The assembly of  claim 38 , wherein the mature islets, the mature EC, or both, are deposited on a scaffold. 
     
     
         41 . The assembly of  claim 40 , wherein the mature islets, mature EC or both, are deposited on the scaffold using a bioink. 
     
     
         42 . The assembly of  claim 41 , wherein the bioink comprises fibrin or alginate.